www.elsevier.com/locate/vetpar

Veterinary Parasitology 146 (2007) 66–76

Combining the effects of supplementary feeding and copper

oxide needles for the control of gastrointestinal

nematodes in browsing goats

C. Martınez Ortiz de Montellano a, J.J. Vargas-Magana b, A.J. Aguilar-Caballero a,C.A. Sandoval-Castro a, L. Cob-Galera a, M. May-Martınez a, R. Miranda-Soberanis a,

H. Hoste c, R. Camara Sarmiento a, J.F.J. Torres-Acosta a,*a Facultad de Medicina Veterinaria y Zootecnia, Universidad Autonoma de Yucatan, Km. 15.5 carretera Merida-Xmatkuil,

Merida, Yucatan, Mexicob Escuela Superior de Ciencias Agropecuarias, Universidad Autonoma de Campeche, Calle 53 s/n X 18,

Col. Unidad Esfuerzo y Trabajo No. 2 Escarcega, Campeche, Mexicoc UMR 1225 INRA/DGER, Ecole Nationale Veterinaire Toulouse, 23 Chemin des Capelles,

31076 Toulouse Cedex, France

Received 10 November 2006; received in revised form 1 February 2007; accepted 14 February 2007

Abstract

The aim was to assess the benefits obtained from combining supplementary feeding and copper needles (COWP), compared

to the use of both approaches independently, for the control of gastrointestinal nematode (GIN) infections in browsing kids.

Forty-four nematode free Criollo kids were exposed to natural parasite infection. The kids were divided into six experimental

groups: not treated, supplemented (NT-S), not treated, not supplemented (NT-NS), moxidectin treated, supplemented (M-S),

moxidectin treated not supplemented (M-NS), copper treated, supplemented (COWP-S) and copper treated, non-supplemented

(COWP-NS). Copper treated groups received Copinox1 (2 g capsules) on day 0 and on day 60 of the trial. Moxidectin treated

groups received Cydectin1 (0.2 mg/kg of body weight sc) every 28 days. Three of the groups received individual

supplementation (100 g of feed/day fresh basis; 74% sorghum: 26% soybean meal; NT-S, M-S and COWP-S) and the other

three groups were not supplemented (NT-NS, M-NS and COWP-NS). Animals browsed native vegetation (6.5 h/day) during

the wet season (154 days). Kids were weighed every 14 days to determine live weight gain (LWG) and blood and faecal

samples were obtained to determine packed cell volume (PCV), haemoglobin (Hb), peripheral eosinophil counts (PEC) and

faecal egg counts (FEC). At the end of the trial, four kids of each group were euthanatized (six kids in each COWP treated

group). Worm burdens, female worm lengths and prolificacy were determined. Liver samples were used to determine copper

concentration and were stained with haematoxylin–eosin to determine microscopic lesions. Animals receiving the combination

of supplementary feeding and COWP improved their LWG, PCV and Hb to similar levels of animals with suppressive AH

treatment. This was not the case when COWP was used without supplementation. Liver copper concentration in COWP treated

groups increased significantly especially in the COWP-NS kids but this was not associated with liver lesions or clinical signs.

Post-mortem Haemonchus contortus and Trichostrongylus colubriformis worm counts had a tendency to be reduced in the

different groups (66–35% reduction) compared to NT-NS group at the end of the trial (P > 0.05). Also, COWP treatment and/

or supplementation reduced female worm length of T. colubriformis and prolificacy of H. contortus and T. colubriformis. This

study, confirmed the value of nutritional supplementation in the control of GIN in growing kids. The use of COWP in addition

* Corresponding author. Tel.: +52 999 942 3200; fax: +52 999 942 3205.

E-mail address: tacosta@uady.mx (J.F.J. Torres-Acosta).

0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2007.02.012

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–76 67

to supplementation had a limited contribution on the kids’ resilience against GIN. This may be due to the reduced infection of

H. contortus during this trial.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Supplementary feeding; Copper needles; Helminth; Browsing; Goats; Resilience

1. Introduction

Due to the constant development of anthelmintic (AH)

resistance in worm populations, the control of gastro-

intestinal nematodes (GIN) in livestock is now moving

from simply relying on drug treatments into a more

complex setting where several approaches are used

together. For this combination to be of any use for

commercial or resource-poor farmers, the benefits from

the interventions must outweigh the cost (Krecek and

Waller, 2006). Supplementary feeding is a good

alternative method for the control of GIN in goats both

in pen trials (Blackburn et al., 1991, 1992) as well as in

field trials (Faye et al., 2003). Field trials with goats in

Mexico showed that supplementary feeding was able to

improve resilience in an economically feasible manner

(Torres-Acosta et al., 2004, 2006). Supplementary

feeding may contribute to reduced economic losses

due to parasitism and, in some cases, to reduce pasture

infectivity (Ketzis et al., 2006). However, supplemented

infected animals still show a reduction in growth

compared to worm free supplemented kids. Therefore,

there is still room for improving the outcome of

supplementary feeding by combining it with another

strategy. Copper oxide wire particles (COWP) capsules

can be a suitable candidate to improve resilience of

naturally infected supplemented kids in tropical regions.

Recent work has shown that COWP capsules adminis-

tered orally to goats have an anthelmintic effect against

Haemonchus contortus with extended protection for up

to 8 weeks (Chartier et al., 2000; Waruiru et al., 2004). If

COWP can effectively control H. contortus for a

sufficient period of the wet season, goats may use the

extra nutrients from supplementary feeding for growth

(resilience) and/or resistance against the other worm

species found in their gastrointestinal tract. Thus, the

economic benefit of supplementary feeding might be

higher. However, the use of COWP might be limited by

the risk of potential copper toxicity (Waller et al., 2004).

The interaction between COWP and supplementary

feeding for the control of GIN in goats has not been

explored. The objective was to compare how beneficial

can be supplementary feeding and COWP combined, as

opposed to both approaches independently, in the control

of GIN infections of browsing kids.

2. Materials and methods

2.1. Location of the study

This study was performed at the FMVZ-UADY

(208520–198300 North, 908000–888500 West) during

the wet season (July–November 2002) for a total of

154 days.

2.2. Experimental animals

Forty-four worm free Criollo kids (8.94 � 1.42 kg

LW) were used from weaning (3 months of age).

2.3. Experimental groups

Animals were randomly distributed in six groups

balanced by weight.

� T

wo naturally infected groups:

� NT-S: Seven kids infected with GIN and supple-

mented.

� NT-NS: Ten kids infected with GIN and non-

supplemented.

� T

wo groups with controlled infection (treated with

injectable moxidectin):

� M-S: Seven kids treated with moxidectin and

supplemented.

� M-NS: Seven kids treated with moxidectin and non-

supplemented.

� T

wo groups with partial control of infection (treated

with copper needles):

� COWP-S: Six kids partially infected and supple-

mented.

� COWP-NS: Six kids partially infected and non-

supplemented.

2.4. Supplementary feeding

Supplemented animals received 100 g sorghum/

soybean meal (74:26) per day. Supplement provided

87.9 g of DM, 11.1 g of metabolizable protein and

0.27 MCal of metabolizable energy. Supplement was

provided in individual cages in the afternoon when the

animals returned from browsing.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–7668

2.5. Anthelmintic treatment

Animals received moxidectin treatment (Cydectin1,

Fort Dodge; 0.2 mg per kg BW s.c.) on day 0 and every

28 days during the experimental period.

2.6. Copper oxide wire particles (COWP)

Animals received COWP (Copinox1, Bayer; 2 g

capsules equivalent to 1.7 metallic copper) on day 0 and

day 60 of the trial per os. Dosing took place in the

afternoon. Animals were kept in individual cages after

dosing and animals were supervised during the after-

noon and the following morning in order to confirm that

capsules were not rejected. This dosing strategy was

based on previous epidemiological information which

indicated that H. contortus infectivity of the browsing

area was high from July to October (Torres-Acosta

et al., 2004). The use of COWP on day 0 aimed at

controlling H. contortus from the beginning of possible

challenge, avoiding possible inefficacy of COWP as an

AH due to an increased abomasal pH of kids (Bang

et al., 1990a).

2.7. Browsing management

Animals browsed daily in a native deciduous

tropical forest for 6.5 h every day (7:00–13:30 h).

The forest contained mainly forage trees and bushes

together with a small proportion of native grasses. The

kids browsed together with a herd of 300 naturally

infected adult goats in order to contribute to the

contamination of the browsing area during the

experimental period. This management was meant to

offer a continuous infection challenge for the experi-

mental animals. A goat keeper took all the animals to

the browsing area every day.

2.8. Paddock infectivity

Infectivity of the browse was monitored for the

duration of the trial by means of consecutive pairs of

tracer kids which browsed for a period of 30 days after

which they were kept in pens with concrete floor for 21

days before being humanly slaughtered.

2.9. Live weight gain

All the kids were weighed on a calibrated scale and

sampled on day 0 and every 14 days. Weighing and

samplings took place at 7:00 a.m. 16 h after their last

daily meal.

2.10. Haematological techniques

At each sampling point animals were sampled 3 ml

of blood from the jugular vein of each kid using tubes

previously identified. Blood samples were processed for

packed cell volume (PCV), haemoglobine (Hb) and the

peripheral eosinophil counts (PEC) as described by

Torres-Acosta et al. (2004).

2.11. Parasitological techniques

Individual faecal samples were collected (3 g faeces/

animal) directly from the rectum to determine the faecal

egg counts (FEC) by means of a modified McMaster

technique with a sensitivity of 50 eggs/g (EPG)

(Withlock, 1948). Every 28 days faeces were pooled

for each experimental group to identify the genus of

infective larvae (days 28, 56, 84, 112 and 140 of the

trial). Each animal in the experimental groups

contributed with 1 g of faeces for the bulk culture.

Faeces were gently mixed, pellets were broken and

placed in a Petri dish. The bulk faecal culture was

placed inside a bigger Petri dish containing a thin layer

of water to keep the culture moisten. Every second day

the culture was aerated and misted with water. Third

stage larvae were collected from the group faecal

cultures after 7 days using the Corticelli–Lai technique

(Corticelli and Lai, 1963). The genera of GIN present in

the cultures were identified using adequate keys of

identification (Bowman and Lynn, 1999). The purpose

of producing pooled faecal cultures per group every 28

days was to monitor indirectly the tendency of

nematode genus present in the experimental animals

on different windows of time during the trial (Burke

et al., 2005a).

2.12. Post-mortem studies

At the end of the trial (day 154), four animals per

group were humanly slaughtered. The abomasums,

small intestine and large intestine were ligated and

removed from the animal. The organs were processed

separately immediately after slaughter. In the COWP

treated groups all the animals were slaughtered. This

took place 94 days after the last dose with COWP.

2.13. Worm count and worm identification

Worm counts (adults and immature adults) were

determined in 10% aliquots from the contents and

washings obtained from the abomasum, small intestine

and large intestine separately as described by MAFF

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–76 69

(1986). In order to assure total worm recovery, each

aliquot was screened twice by two trained workers.

Worm identification was made using all the specimens

found in the aliquots obtained from the abomasum,

small and large intestine, respectively using the keys of

identifications of Barth and Visser (1991).

2.14. Length of female worms

Up to 40 adult female worms from each section of

the gastrointestinal tract (abomasums, small and large

intestines) of each slaughtered kid were measured using

a calibrated grid in a stereo microscope (4�).

2.15. Number of eggs in utero of female worms

Previously identified adult female H. contortus and

Oesophagostomum columbianum were placed in

Eppendorf tubes and were ground with a rod to release

the eggs from the uterus. The tube was filled with 1 ml

of water and a 0.1 ml aliquot withdrawn using a 1 ml

syringe. This was placed in a microscope slide (five

drops) and the number of eggs was determined at 10�magnification. Eggs counted in the five drops were

added and then multiplied by 10 to give the total number

of eggs. The number of eggs in the uterus of

Trichostrongylus colubriformis females was directly

observed in the microscope (40�).

2.16. Number of histotropic or inhibited larvae

The abomasum, small and large intestines of

slaugthered kids were artificially digested separately

using the technique described by MAFF (1986). The

number of histotropic or inhibited larvae (fourth-stage

larvae) was counted from 10% aliquots (20 ml each)

from the digestion material of abomasums, small and

large intestines, respectively. In order to assure total

larvae recovery, each aliquot was screened twice by two

trained workers. Aliquots were coloured with iodine

before being examined for worms.

2.17. Histopathology of the liver and liver copper

concentration

Two samples were obtained from the liver of the

slaughtered kids. One tissue sample was preserved in

10% neutral buffered formalin (pH 7.4) and dehy-

drated through graded alcohols before being

embedded in paraffin wax, in accordance to standard

procedures (Luna, 1965). Sections (5 mm) were

stained with haematoxylin and eosin (HE) for

histopathological examination. A second sample

(10 g fresh tissue) was used to determine the

concentration of copper with a colorimetric spectro-

photometer at 440 NM (FMVZ-UADY Diagnostic

Laboratory, Merida, Yucatan).

2.18. Data analysis

PCV, Hb, PEC and EPG, were analyzed with

repeated measures ANOVA of the general linear

models (GLM) using the SPSS software. The model

used treatment, day, treatment by day and a repeated

statement for day of measurement. PEC and EPG

transformed to log 10 (value+1) before their respec-

tive analysis. The EPG data analysis included from

day 28 of the trial, when infection was patent in all

the groups. In the variables where the null hypothesis

was rejected a tukey post-hoc analysis was per-

formed. The mean total LWG of each group were

compared with the ANOVA test with the tukey post-

hoc test.

The post-mortem variables (number of worms,

number of eggs in utero and female worm length)

were checked for normality by means of a Kolmo-

gorov–Smirnov test for each worm species (H.

contortus, T. colubriformis, and O. columbianum).

Variables that normally distributed were analyzed

using a GLM procedure and their respective tukey

post-hoc tests. Variables that were not normally

distributed were transformed by means of the Box–

Cox procedure. Variables that were not normally

distributed, even after transformation, were compared

using the Kruskal–Wallis tests, one for each worm

species. When a Kruskal–Wallis test was significant,

Wilcoxon rank sum tests were used to determine

which pairs of groups differ (Petrie and Watson,

2006). Only adult worms were included in the

analysis of female worm lengths and the number of

eggs in utero.

Data of Cu concentration (ppm fresh tissue)

determined in the liver of kids of the NT-S, COWP-S

and COWP-NS groups were analyzed by ANOVA using

GLM procedure and the tukey post-hoc test. The

frequency of lesions in the liver, determined by

histopathology, was compared between groups using

Fisher exact tests (EPIinfo v 6.0).

Costs (COWP capsules, food and AH treatment) and

incomes (LWG) from the different groups were

recorded and a partial budget analysis performed as

described by Torres-Acosta et al. (2004). All the

comparisons were made against the costs and incomes

of the NT-NS kids.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–7670

Table 1

Effect of supplementary feeding and/or copper oxide wire particles

(COWP) on the mean total liveweight gain (kg) of Criollo kids

naturally infected with GIN in tropical Mexico

Supplementation Worm control Live weight gain

(mean � S.E.)*

S Moxidectin (M) 7.86 � 0.54 a

NS Moxidectin (M) 5.47 � 0.47 b

S Not-treated (NT) 4.94 � 0.51 b

NS Not-treated (NT) 1.90 � 0.54 c

S COWP 7.16 � 0.57 ab

NS COWP 1.27 � 0.52 c

* Means with different letters (a–c) in a column indicate significant

difference (P < 0.05).

3. Results

3.1. Rainfall pattern during the trial

The total amount of rain recorded during this trial was

880 mm. A hurricane (Isidore) hit the study area on the

22nd of September (day 80 of the trial). As a result the

amount of rain recorded around that period accounted for

almost half of the total rainfall during the study period. In

the months of October and November the amount of

rainfall was negligible. One animal in the COWP-S group

died during the hurricane due to asphyxia.

3.2. Infectivity of the browsing area

Consecutive pairs of tracer kids showed that the

browsing area had a fluctuating infectivity which

included larvae of H. contortus, T. colubriformis and

O. columbianum (Fig. 1). Mean H. contortus burden of

the tracer kids was lower than 500 adult worms during

the study except in September when worm burdens

average 2000. T. colubriformis were consistently more

abundant in the tracer kids with the exception of

September. O. columbianum worm burdens of tracer

kids were low for the duration of the trial.

3.3. Live weight gain

The combination of supplementary feeding and

COWP (COWP-S) helped to achieve similar total

LWG than that of the group maintained free of infection

(M-S) (Table 1 and Fig. 2). Meanwhile, the use of

supplementation only (NT-S) did not show the same

degree of effectiveness and LWG was smaller than that

of M-S group. Although COWP-S kids showed a

tendency to have higher LWG than NT-S kids

Fig. 1. Mean worm burdens from pairs of tracer kids corresponding to

the month when they browsed during the trial. Hc: Haemonchus

contortus; Tc: Trichostrongylus colubriformis; Oc: Oesophagosto-

mum columbianum.

(7.16 � 0.57 versus 4.94 � 0.51), this was not sig-

nificantly different probably due to sample size.

Another important finding is that the use of COWP

alone (COWP-NS group) did not have a positive impact

on growth compared to the infected non-supplemented

group (P > 0.05).

Fig. 2. Effect of supplementation (S) and copper oxide wire particles

(COWP) on the CLWG, PVC and HB in Criollo goats.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–76 71

3.4. Haematological values

The NT-NS group had lower PCVand Hb compared to

that of the M-S, NT-S and COWP-S groups (P < 0.05;

Fig. 2). Although the mean PCV and the Hb of the

COWP-NS group seemed to be lower than that of the

M-S, NT-S and COWP-S groups, the difference was not

significant. Furthermore, there was no difference

between groups in the PEC.

3.5. Worm egg counts and faecal cultures

Faecal egg counts of the different infected groups

(NT-S, NT-NS, COWP-S and COWP-NS) were similar

for the duration of the trial (P < 0.05; Fig. 3).

Faecal cultures on day 28 were not made as most

experimental animals started shedding nematode eggs

after that time. Faecal cultures showed that on day 56 of

the trial (end of August) the faeces of the infected groups

(NT-S, NT-NS, COWP-S and COWP-NS) had larvae of

Haemonchus spp., Trichostrongylus spp. and Oesopha-

gostomum spp. On day 84 of the trial (end of September),

the groups treated with COWP had mostly Trichos-

trongylus spp. larvae and the NT-S and NT-NS groups

had Haemonchus spp., Trichsotrongylus spp. and

Oesophagostomum spp. On day 112 (end of October),

the faecal culture of the COWP treated groups had mostly

Oesophagostomum spp., a little proportion of Trichos-

trongylus spp., and even fewer Haemonchus spp. Finally,

on day 140 (end of November) all the infected groups had

similar quantities of larvae of the three nematode species.

3.6. Worm burdens

Slaughtered animals from the moxidectin treatment

(M-S and M-NS) had no worms at the end of the trial

Fig. 3. Effect of supplementation (S) and copper oxide wire particles

(COWP) on the nematode eggs per gram of faeces (EPG) of Criollo

goats.

(17 days after the last moxidectin treatment). Worm

burdens of infected groups (median, range, mean and

standard error of the mean) are shown in Table 2.

There were no significant differences between worm

burdens of the different groups. However, mean adult

H. contortus and T. colubriformis worms (male, female

and total) in the NT-S and the COWP-S groups were one

third or smaller than that of NT-NS group. Slaughter

took place 94 days after last dose with COWP.

3.7. Female worm length and eggs in uterus

Female worm lengths and the number of eggs in

utero of H. contortus, T. colubriformis, O. columbianum

are shown in Table 3. Female worm length of

T. colubriformis was larger in the NT-NS group

compared to that of copper treated and/or supplemented

groups (P < 0.05). A similar tendency was found in the

H. contortus females (P > 0.05). Copper treatment and/

or supplementary feeding induced a reduction in the

number of eggs in utero of the H. contortus and

T. colubriformis female worms compared to NT-NS

(P < 0.05).

3.8. Copper concentration in the liver

Both COWP treated groups had higher copper

concentration in the liver than that of the control

animals (43.6 � 5.9 ppm fresh weight; n = 8;

P < 0.05). However, kids in the COWP-NS group

had a higher mean Cu concentration in the liver

(103.7 � 6.9 mg/kg fresh weight; n = 6) compared to

the kids in the COWP-S group (66.8 � 7.5 mg/kg fresh

weight; n = 5; P < 0.05).

3.9. Histopathological findings

The different experimental groups had similar

proportions of animals with microscopic lesions in

the liver (P > 0.05). Lesions did not show any

association with copper treatment. The most frequent

lesions observed in the liver of the animals were

lipidosis, necrosis, cholestasis and tumefaction. Also,

several kids showed regeneration processes.

3.10. Economic analysis

The use of supplementary feeding increased eco-

nomic income by 2.9 USD per animal compared to NT-

NS kids. However, the combination of supplementary

feeding and COWP doubled the income per animal

reaching 6.0 USD. This result was close to that of

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Table 2

Effect of supplementary feeding and/or copper oxide wire particles (COWP) on the worm burdens of Criollo kids browsing in tropical Mexico

Worm species Stage NT-S (n = 4) NT-NS (n = 4) COWP-S (n = 6) COWP-NS (n = 6)

Median (range) Mean � S.E.M. Median (range) Mean � S.E.M. Median range Mean � S.E.M. Median range Mean � S.E.M.

H. contortus Larvae 270 (80–560) 303.0 � 241.7 415 (80–750) 415.0 � 473.8 200 (70–680) 200.0 � 251.1 375 (240–570) 375.0 � 155.9

Juvenile 91 (40–390) 153.0 � 54.0 0 (0–0) 0.0 � 54.0 75 (10–300) 103.3 � 44.1 165 (0–240) 131.7 � 44.1

Male 315 (200–1540) 592.5 � 296.7 1005 (370–1860) 1060.0 � 296.7 215 (0–1500) 488.3 � 242.2 595 (250–1500) 685.0 � 242.2

Female 445 (190–1660) 685.0 � 349.1 1360 (370–2300) 1347.0 � 349.1 320 (0–1600) 541.7 � 285.0 675 (270–1720) 808.3 � 285.0

Total 816 (340–1900) 995.0 � 530.3 2365 (340–4000) 2885.0 � 530.3 610 (30–3100) 1531.7 � 433.0 1480 (1260–2340) 1880.0 � 433.0

T. colubriformis Larvae 0 (0–10) 3.3 � 5.8 a 215 (150–280) 215.0 � 91.9 b 40 (0–80) 38.0 � 33.5 a 30 (20–90) 41.7 � 28.6 ab

Juvenile 95 (20–230) 110.0 � 19.5 a 15 (0–30) 15.0 � 19.5 b 0 (0–0) 0.0 � 15.9 c 0 (0–0) 0.0 � 15.9 c

Male 755 (460–2400) 1092.5 � 449.0 3010 (560–3200) 2445.0 � 449.0 1315 (20–2290) 1285.0 � 366.6 1630 (830–2400) 1608.3 � 366.6

Female 870 (340–1900) 995.0 � 530.3 3600 (340–4000) 2885.0 � 530.3 1430 (30–3100) 1531.7 � 433.0 1930 (1260–2340) 1880.0 � 433.0

Total 1640 (1100–4410) 2197.5 � 962.1 6625 (930–7200) 5345.0 � 962.1 2745 (50–5300) 2816.7 � 785.5 3470 (2090–4700) 3488.3 � 785.5

O. columbianum Larvae 6 (0–60) 6.0 � 33.0 ab 155 (150–160) 155.0 � 7.1 b 40 (10–110) 40.0 � 41 ab 10 (0–40) 10.0 � 15.1 a

Juvenile 15 (4–30) 16.0 � 11.2 0 (0–50) 12.5 � 11.2 20 (0–40) 20.3 � 9.1 15 (10–80) 29.2 � 9.1

Male 65 (13–150) 73.3 � 17.9 20 (0–100) 35.0 � 17.9 20 (0–50) 21.7 � 14.6 75 (40–90) 67.5 � 14.6

Female 65 (3–190) 80.8 � 24.3 20 (10–100) 37.5 � 24.3 30 (0–90) 35.0 � 19.9 45 (20–110) 56.7 � 19.9

Total 145 (20–370) 170.0 � 46.9 40 (10–250) 85.0 � 46.9 66 (30–150) 77.0 � 38.3 130 (90–280) 153.3 � 38.3

Means with different letters (a–c) in the same line between columns indicate significant difference (P < 0.05); S.E.M., standard error of the means; NT-S, not treated—supplemented; NT-NS, not

treated—not supplemented; COWP-S, copper oxide wire particles—supplemented; COWP-NS, copper oxide wire particles—not supplemented.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–76 73

Table 3

Female worm length and number of eggs in utero of H. contortus, T. colubriformis and O. columbianum in browsing Criollo kids supplemented and/

or treated with copper oxide wire particles (COWP) during the wet season

Nematode species Groups n Female worm length* Eggs in utero*

Median (range) Mean � S.E.M. Median (range) Mean � S.E.M.

H. contortus COWP-NS 138 17 (11–22) 17.1 � 0.19 a 130 (20–490) 132.9 � 8.05 a

COWP-S 97 18 (15–22) 18.1 � 0.23 b 120 (30–400) 130.4 � 9.60 a

NT-NS 87 20 (10–24) 18.6 � 0.25 b 190 (30–610) 221.7 � 10.14 c

NT-S 97 18 (12–22) 18.1 � 0.23 b 140 (20–610) 178.6 � 9.60 b

T. colubriformis COWP-NS 231 7 (6.0–9.0) 7.2 � 0.04 a 14 (2–30) 14.1 � 0.33 a

COWP-S 171 7 (5.0–9.0) 7.2 � 0.05 a 15 (4–33) 14.8 � 0.39 a

NT-NS 98 8 (6.0–10.0) 7.7 � 0.06 b 17 (3–32) 16.9 � 0.51 b

NT-S 107 7 (5.5–8.5) 7.0 � 0.06 a 10 (1–23) 10.6 � 0.49 c

O. columbianum COWP-NS 28 17.0 (9–19) a 15.1 � 0.57 130 (60–700) 223.2 � 30.75 a

COWP-S 28 17.5 (11–20) a 16.6 � 0.57 105 (10–400) 137.1 � 30.75 a

NT-NS 18 17.0 (9–19) a 15.4 � 0.71 150 (20–500) 162.2 � 38.36 a

NT-S 14 16.0 (9–20) a 15.6 � 0.81 435 (130–790) 427.9 � 43.49 c

NT-S, not treated—supplemented; NT-NS, not treated—not supplemented; COWP-S, copper oxide wire particles—supplemented; COWP-NS,

copper oxide wire particles—not supplemented.* Different letters (a–c) in the same column of the same worm species represents significant differences (P < 0.05).

animals kept under suppressive AH treatment and

supplemented (6.7 USD per animal).

4. Discussion

This trial explored the potential benefit of combining

two alternative methods for the control of GIN. The

rational behind this attempt was to control H. contortus,

one of three worm species common to goats under

browsing conditions of Yucatan, and leave the animals

to defend themselves against the other two important

worm species in the area: T. colubriformis and

O. columbianum (Torres-Acosta et al., 2004, 2006).

H. contortus is considered to be the most pathogenic

parasite of small ruminants on a worm-for-worm basis

(Krecek and Waller, 2006). H. contortus population was

meant to be partially and temporarily under control for a

considerable proportion of the trial by dosing worm free

kids with one 2 g capsule of COWP on day 0 and a

second 2 g capsule on day 60 of the trial. COWP has

being proven to be an efficacious non-conventional

anthelmintic particularly against H. contortus in sheep

(Bang et al., 1990b; Goncalves and Menezes, 2004;

Burke et al., 2004, 2005a) and goats (Chartier et al.,

2000). By using COWP to control H. contortus, treated

kids were expected to have an additional improvement

on their resilience and/or resistance from supplemen-

tary feeding against the other two species of GIN. In this

trial, H. contortus infectivity of the browsing area was

mild (according to tracer kids). Thus the potential

benefit might have been limited. In spite of that, the

results are encouraging since the combination of COWP

and supplementary feeding showed a tendency to

improve LWG in the browsing kids compared to the use

of supplementation alone (P > 0.05). This combination

of supplementary feeding and COWP treatment may

have allowed the kids to take advantage of the time

when H. contortus was under some level of control

improving their growth rate and blood parameters

(improved resilience).

This trial also showed that the sole use of COWP

(COWP-NS) was less effective for improving growth of

kids than the sole use of suppressive AH treatment

(moxidectin treatment every 28 days) (M-NS). Further-

more, the sole use of COWP did not improve LWG or

haematological values compared to NT-NS kids.

The COWP treated groups did not show a reduction

in FEC during this trial. The low efficacy may be due to

the low proportions of H. contortus compared to other

worm species as reported by Burke et al. (2005b) and

Burke and Miller (2006). In this trial, tracer kids showed

that infectivity with H. contortus was lower than that of

T. colubriformis for most of the trial (except for

September). Previous epidemiological information

suggested a higher H. contortus infectivity than that

of T. colubriformis from August and reduced gradually

from October onwards (Torres-Acosta et al., 2004).

However, results of tracer kids showed that the

hurricane which hit the site of the study, Yucatan, at

the end of September, reduced the infectivity of the

browse during October possibly affecting the pattern of

infectivity in the last months of the wet season.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–7674

Even when FEC were not reduced by COWP, the

faecal cultures showed that H. contortus larvae were

present in COWP treated groups on day 56 post-copper

treatment, time when this copper was not expected to

have an effect. The following faecal cultures of COWP

treated groups (24 and 52 days after the 2nd COWP

treatment) showed negligible levels of H. contortus

larvae. It is likely that cultures of day 24 post-COWP

treatment show an effect of copper on the H. contortus

population in these experimental groups. However,

on day 52 post-second treatment the low levels of

H. contortus may only indicate that re-infection did not

occurred. Finally, in the faecal cultures of November

(94 days after 2nd COWP treatment), there were

H. contortus larvae in all the experimental groups. The

reduction in H. contortus cultured in faeces from

COWP-treated groups has been reported earlier in sheep

(Burke et al., 2005a).

Worm burdens at the end of the trial (day 154)

were not significantly different between infected

groups. This was expected as the last COWP

treatment was performed 94 days before slaughter

and the persistent effect reported for COWP against

H. contortus in sheep varies from 21 to 42 days (Bang

et al., 1990b; Knox, 2002; Goncalves and Menezes,

2004). Animals in this trial received a constant

natural infection which is different to the experi-

mental protocols used previously elsewhere. Thus,

any anthelmintic effect in the first days after COWP

treatment may be masked by further establishment of

incoming L3 larvae in the following weeks post-

COWP treatment (Waller et al., 2004). However,

despite the lack of statistical differences, the

following tendencies can still be suggested. The

supplementation was associated with substantial

reduction in H. contortus and T. colubriformis

populations, associated with a decreased fecundity

in both worm species and a reduced female length in

the last one. This confirms previous results obtained

with naturally infected Criollo kids under browsing

conditions (Gutierrez-Segura et al., 2003). On the

other hand, the use of COWP alone also showed

similar effects on worm burdens of H. contortus and

even T. colubriformis. This non-significant reduction

in H. contortus worm burdens was similar to that

reported in trials were H. contortus was trickle

infected in goats (Chartier et al., 2000; 39.2% on day

70 post-COWP treatment, P > 0.05) and also in sheep

(Knox, 2002; 54% on day 42 post-COWP treatment,

P = 0.058). Although kids in the COWP-S treated

groups tended to have nearly one third of the H.

contortus and T. colubriformis worm burdens of the

NT-NS kids, the combination of COWP and

supplementation did not appear to add much

compared to supplementation alone in regard of

effects on biology of worm populations although the

benefit for resilience has been previously underlined.

Last, for O. columbianum populations, whatever the

treatment, no effects were found.

Copper levels in the liver of browsing kids without

COWP treatment were within normal levels of goats

and sheep receiving no copper supplementation in

other latitudes (Glennon et al., 2004; Waller et al.,

2004). This is consistent with findings of Millan-

Cortes et al. (1990) who reported that grass of the

region have sufficient copper for ruminant nutrition

(8–10 ppm) although it can reach levels of deficiency

during the wet season. COWP treated groups (COWP-

S and COWP-NS) increased their copper concentra-

tion in the liver, being significantly higher in COWP-

NS. This increment of copper concentration in the

liver due to COWP treatment has been reported in

sheep (Bang et al., 1990b; Goncalves and Menezes,

2004; Waller et al., 2004) and goats (Chartier et al.,

2000; Glennon et al., 2004). However, no overt signs

of copper toxicity have been found in previous trials

and the same is true for the present study.

Interestingly, we found evidence indicating that

copper liver concentration in COWP treated kids

depended on the availability of supplementary

feeding contrary to early evidence with sheep (Burke

et al., 2004). It is possible that the higher copper

concentration in the COWP-NS kids resulted from

their low growth rate compared to COWP-S. Both

groups were exposed to the same amount of copper

but the faster growing COWP-S kids might have used

more copper for their metabolic processes such as

erythropoiesis, protection from oxidants, develop-

ment of heart and central nervous system, immuno-

competence (Underwood and Suttle, 2001) than the

COWP-NS kids. It is important to point out that using

COWP without supplementary feeding increases the

risk of chronic copper toxicity compared to supple-

mented kids even when animals are eating in the same

browsing area.

This trial showed that the use of supplementation

may be more reliable and easy to implement than

COWP (either combined or alone). This trial showed

that COWP treatment cannot be widely recommended

for the control of GIN in tropical farming systems

because it is difficult to foresee the H. contortus

challenge that animals will have before and after COWP

treatment. In addition, the risk of chronic copper

toxicity exists.

C.M.O. de Montellano et al. / Veterinary Parasitology 146 (2007) 66–76 75

5. Conclusions

This study, confirmed the value of nutritional

supplementation in the control of GIN in growing

kids. The use of COWP in addition to supplementation

had a limited contribution on the kids’ resilience against

GIN. This may be due to the reduced infection of H.

contortus during this trial. Thus, relaying on COWP

treatment alone was ineffective. An accumulation of

copper in the liver COWP treated animals was observed,

thus veterinary advice is crucial to limit chronic toxicity

problems.

Acknowledgments

This work was financially supported by the

Universidad Autonoma de Yucatan, Mexico (project

PRIORI-FMVZ 02-011) and ECOS-Nord (France)/

ANUIES-CONACyT (Mexico) (project M03-A03). We

thank Animax Ltd. for providing the COWP capsules

for this trial.

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